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The Butyrivibrio fibrisolvens tet(W) Gene Is Carried on the Novel Conjugative Transposon TnB1230, Which Contains Duplicated Nitroreductase Coding Sequences. Claire M. Melville, 2004.The Butyrivibrio fibrisolvens tet(W) gene is located on the conjugative transposon TnB1230 . TnB1230 encodes transfer proteins with 48 to 67% identity to some of those encoded by Tn1549 . tet(W) is flanked by directly repeated sequences with significant homology to oxygen-insensitive nitroreductases . The 340 nucleotides upstream of tet(W) are strongly conserved and are required for tetracycline resistance . Comparative Proteome Analysis of Brucella melitensis Vaccine Strain Rev 1 and a Virulent Strain, 16M. Michel Eschenbrenner, 2002.The genus Brucella consists of bacterial pathogens that cause brucellosis, a major zoonotic disease characterized by undulant fever and neurological disorders in humans . Among the different Brucella species, Brucella melitensis is considered the most virulent . Despite successful use in animals, the vaccine strains remain infectious for humans . To understand the mechanism of virulence in B . melitensis, the proteome of vaccine strain Rev 1 was analyzed by two-dimensional gel electrophoresis and compared to that of virulent strain 16M . The two strains were grown under identical laboratory conditions . Computer-assisted analysis of the two B . melitensis proteomes revealed proteins expressed in either 16M or Rev 1, as well as up- or down-regulation of proteins specific for each of these strains . These proteins were identified by peptide mass fingerprinting . It was found that certain metabolic pathways may be deregulated in Rev 1 . Expression of an immunogenic 31-kDa outer membrane protein, proteins utilized for iron acquisition, and those that play a role in sugar binding, lipid degradation, and amino acid binding was altered in Rev 1 . Overproduction of Inactive Variants of the Murein Synthase PBP1B Causes Lysis in Escherichia coli. Ute Meisel, 2003.Penicillin-binding protein 1B (PBP1B) of Escherichia coli is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain . The protein is present in three forms (  , ß, and
 ) which differ in the length of their N-terminal cytoplasmic region . Expression plasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed . The inactive domains contained a single amino acid exchange in an essential active-site residue . Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells . The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions . Lysis was also reduced in mutant strains lacking several murein hydrolases . In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lytic transglycosylases are responsible for the observed lysis effect . A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a multienzyme complex is discussed .
Production of Cyclic Lipopeptides by Pseudomonas fluorescens Strains in Bulk Soil and in the Sugar Beet Rhizosphere. Tommy Harder Nielsen, 2003.The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere . Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca . 5 µg g-1) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil . When a whole-cell inoculum of P . fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week . By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil . In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for  2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days . All three CLPs remained detectable for several days in the rhizosphere . Subsequent tests of five other CLP-producing P . fluorescens strains also demonstrated significant production in the young rhizosphere . The results thus provide evidence that production of different CLPs is a common trait among many P . fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil .
Optimization of a Reusable Hollow-Fiber Ultrafilter for Simultaneous Concentration of Enteric Bacteria, Protozoa, and Viruses from Water. Hugo A. Morales-Morales, 2003.The detection and identification of pathogens from water samples remain challenging due to variations in recovery rates and the cost of procedures . Ultrafiltration offers the possibility to concentrate viral, bacterial, and protozoan organisms in a single process by using size-exclusion-based filtration . In this study, two hollow-fiber ultrafilters with 50,000-molecular-weight cutoffs were evaluated to concentrate microorganisms from 2- and 10-liter water samples . When known quantities (105 to 106 CFU/liter) of two species of enteric bacteria were introduced and concentrated from 2 liters of sterile water, the addition of 0.1% Tween 80 increased Escherichia coli strain K-12 recoveries from 70 to 84% and Salmonella enterica serovar Enteritidis recoveries from 36 to 72% . An E . coli antibiotic-resistant strain, XL1-Blue, was recovered at a level (87%) similar to that for strain K-12 (96%) from 10 liters of sterile water . When E . coli XL1-Blue was introduced into 10 liters of nonsterile Rio Grande water with higher turbidity levels (23 to 29 nephelometric turbidity units) at two inoculum levels (9 x 105 and 2.4 x 103 per liter), the recovery efficiencies were 89 and 92%, respectively . The simultaneous addition of E . coli XL1-Blue (9 x 105 CFU/liter), Cryptosporidium parvum oocysts (10 oocysts/liter), phage T1 (105 PFU/liter), and phage PP7 (105 PFU/liter) to 10 liters of Rio Grande surface water resulted in mean recoveries of 96, 54, 59, and 46%, respectively . Using a variety of surface waters from around the United States, we obtained recovery efficiencies for bacteria and viruses that were similar to those observed with the Rio Grande samples, but recovery of Cryptosporidium oocysts was decreased, averaging 32% (the site of collection of these samples had previously been identified as problematic for oocyst recovery) . Results indicate that the use of ultrafiltration for simultaneous recovery of bacterial, viral, and protozoan pathogens from variable surface waters is ready for field deployment .
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